Hb S polymerization and sickling are the central events in the pathophysiology of sickle cell disease. The blood of these patients is characterized by the presence of dehydrated, dense erythrocytes, with an elevated hemoglobin S concentration. Since increases in hemoglobin S concentration markedly increase the rate of Hb S polymer formation and the extent of cell sickling, a possible therapeutic approach is based on the prevention of cell dehydration by specific blockade of the transport pathways involved. Two K efflux pathways play a major role in cell dehydration, namely the K-C cotransport system and the Ca-activated (Gardos) K channel. The molecular identity of K-Cl cotransport system and the Ca-activated (Gardos) K channel. The molecular identity of K-Cl cotransport (hKCC1) has been recently elucidated. Although the erythroid Gardo channel has not been yet cloned, several related Ca-gated K channels of large (BK) and small (SK) conductance have been cloned. Studies in vitro in sickle erythrocytes and in vivo in transgenic SAD mice and patients with homozygous Hb S (ss) disease have shown that dehydration of sickle erythrocytes can be diminished by specific blockade of the Gardos channel by the imidazole antimycotic clotrimazole (CLT) or by specific blockade of K-Cl cotransport by increasing the erythrocyte microgram content via dietary microgram supplements. Our studies will be aimed at the following: 1) Role of Gardos channel and K-Cl cotransport in dehydration of sickle erythrocytes and reticulocytes; 2) Molecular characterization of the human and mouse erythrocyte K-Cl cotransporters: The major focus of these two specific aims is the molecular and biophysical characterization of the Gardos channel and the K-Cl cotransporter, with special emphasis on those properties which can be therapeutically manipulated in mouse and in humans, as shown in our ongoing clinical and mouse studies. 3) The effect of in vivo modulation of K transport pathways on red cell hydration state and on sickle disease phenotype in transgenic sickle mice: We will focus on how the clinical manifestation of sickle cell disease in the mouse model can be affected by modulating the activity of either one of the two transport pathways. The effect of pharmacological blockade of K-Cl cotransport or Gardos channel will be studied, as ell as the effect of up- and down-modulation of K-Cl cotransport by either breeding sickle mice into strains with high or low K-Cl cotransport activity or by genetic manipulation of K-Cl cotransport gene or its regulators. These studies are designed to gain insight into the bases of cell dehydration in sickle cells with the final objective of developing new therapeutic options of patients with sickle cell disease. They represent the logical extension of the productive collaboration and successful studies supported by the present funding cycle.